Crystals of sarcoplasmic reticulum Ca(2+)-ATPase

High-resolution structures of the Ca(2+)-ATPase have over the last 5 years added a structural dimension to our understanding of the function of this integral membrane protein. The Ca(2+)-ATPase is now by far the membrane protein where the most functionally different conformations have been described in precise structural detail. Here, we review our experience from solving Ca(2+)-ATPase structures: a purification scheme involving minimum handling of the protein to preserve natural and essential lipids, a rational approach to screening for crystals based on a limited number of polyethyleneglycols and many different salts, improving crystal quality using additives, collecting the data and finally solving the structures. We argue that certain of the lessons learned in the present study are very likely to be useful for crystallisation of eukaryotic membrane proteins in general.     

Reconstructing an ultrametric galled phylogenetic network from a distance matrix

Given a distance matrix M that specifies the pairwise evolutionary distances between n species, the phylogenetic tree reconstruction problem asks for an edge-weighted phylogenetic tree that satisfies M, if one exists. We study some extensions of this problem to rooted phylogenetic networks. Our main result is an O(n(2) log n)-time algorithm for determining whether there is an ultrametric galled network that satisfies M, and if so, constructing one. In fact, if such an ultrametric galled network exists, our algorithm is guaranteed to construct one containing the minimum possible number of nodes with more than one parent (hybrid nodes). We also prove that finding a largest possible submatrix M' of M such that there exists an ultrametric galled network that satisfies M' is NP-hard. Furthermore, we show that given an incomplete distance matrix (i.e. where some matrix entries are missing), it is also NP-hard to determine whether there exists an ultrametric galled network which satisfies it.     

Inferring phylogenetic relationships avoiding forbidden rooted triplets

To construct a phylogenetic tree or phylogenetic network for describing the evolutionary history of a set of species is a well-studied problem in computational biology. One previously proposed method to infer a phylogenetic tree/network for a large set of species is by merging a collection of known smaller phylogenetic trees on overlapping sets of species so that no (or as little as possible) branching information is lost. However, little work has been done so far on inferring a phylogenetic tree/network from a specified set of trees when in addition, certain evolutionary relationships among the species are known to be highly unlikely. In this paper, we consider the problem of constructing a phylogenetic tree/network which is consistent with all of the rooted triplets in a given set C and none of the rooted triplets in another given set F. Although NP-hard in the general case, we provide some efficient exact and approximation algorithms for a number of biologically meaningful variants of the problem.     

Sulfates dramatically stabilize a salt-dependent type of glucagon fibrils

Recent work suggests that protein fibrillation mechanisms and the structure of the resulting protein fibrils are very sensitive to environmental conditions such as temperature and ionic strength. Here we report the effect of several inorganic salts on the fibrillation of glucagon. At acidic pH, fibrillation is much less influenced by cations than anions, for which the effects follow the electroselectivity series; e.g., the effect of sulfate is approximately 65-fold higher than that of chloride per mole. Increased salt concentrations generally accelerate fibrillation, but result in formation of an alternate type of fibrils. Stability of these fibrils is highly affected by changes in anion concentration; the apparent melting temperature is increased by approximately 22 degrees C for any 10-fold concentration increase, indicating that the fibrils cannot exist without anions. In contrast, fibrillation under alkaline conditions is more affected by cations than anions. We conclude that ions interact directly as structural ligands with glucagon fibrils where they coordinate charges and assist in formation of new fibrils. As ex vivo amyloid plaques often contain large amounts of highly sulfated organic molecules, the specific effects of sulfate ions on glucagon may have general relevance in the study of amyloidosis and other protein deposition diseases.     

N- and C-terminal hydrophobic patches are involved in fibrillation of glucagon

Recent work suggests that the molecular structure of amyloid-like fibrils is determined by environmental conditions as well as amino acid sequence. To probe the involvement of side chains in fibrillation of the 29-residue hormone glucagon, we have measured fibrillation kinetics of 15 alanine mutants. At acidic pH, all of the mutants are able to form fibrils. However, substitution of hydrophobic residues in the N- and C-termini (in particular Phe6, Tyr10, Val23, and Met27) decelerates fibrillation dramatically. This indicates that the hydrophobicity and/or high beta-sheet propensity of these residues may be important for fibrillation. In contrast, substitution of Leu14 increases fibrillation propensity compared to that of the wild type. Nevertheless, despite identical fibrillation conditions, the thioflavin T and tryptophan fluorescence spectra of fibrils formed by mutants Tyr13, Leu14, and Asp15 are significantly different from those of other mutants, indicating that substitution of these residues may influence not only the fibrillation kinetics and fibril stability but also the preferred final structure of the fibrils that is formed, in line with the general structural polymorphism of glucagon fibrils. In contrast, under alkaline conditions, only a handful of the alanine mutants are capable of forming fibrils, suggesting that more side chains are involved in stabilizing interactions here. In addition, fibrils formed by wild-type glucagon at alkaline pH appear very stable, compared to fibrils formed at acidic pH. This suggests that the distribution of charges determines the number of different fibrillated states available to a peptide, since these can block formation of metastable fibrillated states.     

Aromatic O-glycosylation

Carbohydrates carrying an aromatic aglycon are important natural products and thus key synthetic targets. However, due to the electron-withdrawing properties of aromatic rings, phenols are difficult to glycosylate. This review covers the most common carbohydrate donors used for aromatic O-glycosylation (anomeric acetates, halides, trichloroacetimidates and thioglycosides) as well as some less common donors. The scope of the review is to give practical examples of aromatic O-glycosylations and to offer guidelines for glycosylation of typical aromatic residues. Anomeric acetates or trichloroacetimidates, activated under acidic conditions, are preferred for electron rich aromatic aglycons, while glycosyl halides, activated using basic conditions, are preferred for electron deficient aromatic residues.     

Residues from transmembrane helices 3 and 5 participate in leukotriene B4 binding to BLT1

Leukotrienes are inflammatory mediators that bind to seven transmembrane, G-protein-coupled receptors (GPCRs). Here we examine residues from transmembrane helices 3 and 5 of the leukotriene B4 (LTB4) receptor BLT1 to elucidate how these residues are involved in ligand binding. We have selected these residues on the basis of (1) amino acid sequence analysis, (2) receptor binding and activation studies with a variety of leukotriene-like ligands and recombinant BLT1 receptors, (3) previously published recombinant BLT1 mutants, and (4) a computed model of the active structure of the BLT1 receptor. We propose that LTB4 binds with the polar carboxylate group of LTB4 near the extracellular surface of BLT1 and with the hydrophobic LTB4 tail pointing into the transmembrane regions of the receptor protein. The carboxylate group and the two hydroxyls of LTB4 interact with Arg178 and Glu185 in transmembrane helix 5. Residues from transmembrane helix 3, Val105 and Ile108, also line the pocket deeper inside the receptor. LTB4 is becoming increasingly important as an immunomodulator during a number of pathologies, including atherosclerosis. Detailed information about the LTB4 binding mechanism, and the receptor residues involved, will hopefully aid in the design of new immunomodulatory drugs.     

Effects of vascular occlusion on muscular endurance in dynamic knee extension exercise at different submaximal loads

Strength training with low load under conditions of vascular occlusion has been proposed as an alternative to heavy-resistance exercise in the rehabilitation setting, when large forces acting upon the musculoskeletal system are unwanted. Little is known, however, about the relative intensity at which occlusion of blood flow significantly reduces dynamic muscular endurance and, hence, when it may increase the training effect. The purpose of this study was to investigate endurance during dynamic knee extension at different loads with and without cuff occlusion. Sixteen subjects (20-45 years of age) with strength-training experience were recruited. At 4 test sessions, the subjects performed unilateral knee extensions to failure with and without a pressure cuff around the thigh at 20, 30, 40, and 50% of their 1 repetition maximum (1RM). The pressure cuff was inflated to 200 mm Hg during exercise with occlusion. Significant differences in the number of repetitions performed were found between occluded and nonoccluded conditions for loads of 20, 30, and 40% of 1RM (p < 0.01) but not for the 50% load (p = 0.465). Thus, the application of a pressure cuff around the thigh appears to reduce dynamic knee extension endurance more at a low load than at a moderate load. These results may have implications regarding when it could be useful to apply a tourniquet in order to increase the rate of fatigue and perhaps also the resulting training effect. However, the short- and long-term safety of training under ischemic conditions needs to be addressed in both healthy and less healthy populations. Furthermore, the high acute pain ratings and the delayed-onset muscle soreness associated with this type of training may limit its potential use to highly motivated individuals.     

The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins

Background  

Urine is a desirable material for the diagnosis and classification of diseases because of the convenience of its collection in large amounts; however, all of the urinary proteome catalogs currently being generated have limitations in their depth and confidence of identification. Our laboratory has developed methods for the in-depth characterization of body fluids; these involve a linear ion trap-Fourier transform (LTQ-FT) and a linear ion trap-orbitrap (LTQ-Orbitrap) mass spectrometer. Here we applied these methods to the analysis of the human urinary proteome.